FLAVONOIDS: BIOCHEMISTRY, BIOPHYSICS, MEDICINE - Tarakhovsky Yu. S. - 2013

PART 2. MECHANISMS OF FLAVONOID ACTION

2.2. Antioxidant Properties of Flavonoids

2.2.1. Oxidation-Reduction Reactions

In biological systems, oxidation processes are most commonly accompanied by The addition of oxygen or the removal of hydrogen, resulting in the molecule donating an electron, which corresponds to the definition of "oxidation". These processes underlie numerous metabolic pathways. However, certain agents not directly involved in metabolic reaction chains can initiate the generation of oxidation by-products and cause cellular damage. Such agents are referred to as pro-oxidants. Pro-oxidants include so-called reactive oxygen species (ROS). There are also reactive nitrogen species (RNS), chlorine, sulfur, and phosphorus species.

It should be noted that ROS are constantly generated during normal metabolic processes and, in low concentrations, participate in Cell signaling. Cellular damage occurs only when ROS are produced in excess. Furthermore, cellular damage by ROS agents is not only observed in pathological processes but is also utilized by phagocytes to destroy foreign Proteins and unicellular organisms. Agents that prevent The formation of ROS and thereby protect Cells from damage are called antioxidants.

To maintain normal physiological function, a balance between Oxidation and reduction processes must be preserved within The Cell. This fully applies to maintaining a balanced ratio of antioxidants and ROS in the Organism. The literature frequently discusses issues associated with excessive ROS production leading to so-called oxidative stress, which is often proposed to be "combated" by increasing antioxidant concentrations. However, one should not forget that an excess of reducing agents is equally undesirable and can trigger reductive stress—a condition that is less studied yet demands no less attention from researchers, as it can cause various diseases. For instance, an excessive intracellular content of Glutathione, one of the most critical cytoplasmic reducing agents, can paradoxically exert a toxic effect by activating oxidative processes in the Mitochondria.

2.2.2. Oxidative Stress in Living Systems

Over the two centuries since the discovery of oxygen by Antoine Lavoisier, the need to control the interaction of this agent with the molecular components of living systems has become increasingly urgent. The terms "antioxidants", "oxidative stress", "oxidative damage", and, finally, "free radicals" are frequently used in biology and medicine. The term "free radicals" was first introduced by the American researcher Moses Gomberg. It should be noted that nowadays, in accordance with IUPAC recommendations, the word "free" is considered redundant, since no "non-free" radicals exist. In the mid-1950s, the works of Daniel Gilbert and Rebecca Gersham first put forward the concept regarding The Role of radicals in biological processes leading to organismal Aging.

Numerous studies conducted in recent years have uncovered the mechanisms by which entities containing oxygen, chlorine, sulfur, or nitrogen atoms—elusive by traditional chemical Methods—not only participate in vital functions of living systems, but in some cases act as poisons triggering diverse pathological processes underlying many diseases.

2.2.2.1. Radicals

The most distinctive feature of radicals is their high reactivity. Radicals are defined as atoms, molecules, or ions possessing one or two unpaired electrons in their outer orbital, thereby forming an incomplete electron shell capable of entering into Chemical Reactions. The presence of an unpaired electron in the outer orbital typically confers enhanced reactivity upon the molecule. To form a pair, the molecule tends to capture an additional electron from neighboring molecules, which determines the oxidizing properties of radicals.

The oxygen molecule is one of the primary sources of substance oxidation on Earth. In their ground stable state, oxygen molecules have two unpaired p-orbital electrons at the outer second energy level. This state is called the triplet state and is denoted as ³Σ_g^- (Fig. 39).

Class="center">Fig. 39. Electronic Structure OF THE oxygen atom. The oxygen atom has eight electrons distributed across two energy levels in accordance with the formula 1s²2s²2р⁴. As follows from the formula, on the outer, second level, the p-orbital contains two unpaired electrons capable of participating in chemical reactions and determining an oxygen valence of two.

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Formally, the oxygen molecule can be viewed as a biradical; however, its reactivity is not very high because the electrons of triplet oxygen have identical (parallel) spins (Fig. 40). A substantial increase in reactivity is observed when the oxygen molecule is in an excited singlet state, where the electron spins are antiparallel. Two excited singlet states are known: ¹∆g and ³Σg⁺. Among these, only the ¹∆g state is sufficiently stable and plays a significant role in the oxidation processes of living cells.

Fig. 40. Electronic structure of the p-orbital of the second electron energy level in the oxygen molecule participating in the Formation of the interatomic bond. The triplet (ground) and two singlet (excited) states of the oxygen molecule electrons are shown.

2.2.2.2. Radical Nomenclature

In chemical formulas, an unpaired electron located in an outer orbital is denoted by a superscript dot on the right, followed, when necessary, by the magnitude and sign of the charge, for example, О₂^•- (rather than О₂-•), or РO₃^•2-. If there are multiple unpaired electrons, a number is placed before the dot, and parentheses may be used, for example: О₂²• or NO^(2•)-.

It must be noted that metals, their ions, or complexes containing unpaired electrons should not be considered radicals, and their unpaired electrons are not designated by a dot in the formula since they reside in inner orbitals. However, in cases where a Ligand that is a radical is attached to a metal, the dot representing the radical should be used.

Unbound (free) uncharged atoms are named after the element, which may be preceded by the prefix "mono" if necessary. Two strategies can be used for naming polyatomic radicals: substitutive nomenclature and coordination nomenclature. Coordination nomenclature is more effective for naming small inorganic molecules, whereas substitutive nomenclature is suitable for complex organic molecules.

In coordination nomenclature, the central atom is chosen first, for example, Si, C, Sb, As, P, N, S, I, Br, Cl, O, F. It can also be a group of identical or different atoms, such as N₃^•, Cl₂^•-, HO₃^•, ClOO^•, (СN)₂^•-. For homopolyatomic charged radicals, e.g., О₂^•-, the suffix "ide" is used, while for heteropolyatomic charged radicals, including those containing two different atoms where one is hydrogen (such as (HS)₂^•-), the suffix "ate" is used. The hydrogen atom itself is never chosen as the central atom, but may be considered as an attached atom.

Substitutive nomenclature is based on the hydrides of parent molecules. For instance, for СН₄, NН₃, H₂O, PH₃, and H₂S, the respective names methane, azane, oxidane, phosphane, and sulfane are proposed. Various features or modifications of parent molecules are indicated by suffixes. A radical is denoted by the suffix "-yl". A positive charge is denoted by the suffix "-iumyl", and a negative charge by the suffix "-idyl". For example, to name О₂•^-, one must proceed from the fact that the parent of this substance is dioxidane (hydrogen peroxide), which first lost one hydron (H⁺), yielding dioxidanide, and then lost a second hydron, yielding dioxidanidyl (О₂^•-).

2.2.2.3. Pro-oxidants

Prooxidants include radicals and various non-radical active oxidizing agents (Tables 2 and 3). In addition to oxygen atoms, radicals may contain atoms of chlorine, nitrogen, sulfur, and other elements. Accordingly, a distinction is made between reactive oxygen species, reactive chlorine species, reactive nitrogen species, and so forth.

In nature, radicals are formed As a result of molecular Cleavage under METABOLISM/18.html">The Influence of high temperatures or radiation; however, in living cells, radicals are continuously generated through various Chemical Reactions Involving Enzymes and transition-Metal Ions that can act as electron Donors or acceptors.

Table 2. Examples of certain prooxidants—reactive oxygen, nitrogen, chlorine, sulfur, and phosphorus species that are radicals.

Note. The table presents various nomenclatures and designations proposed by the International Union of Pure and Applied Chemistry (IUPAC), as well as those commonly found in the literature. Number (1) denotes names given according to coordination nomenclature, and number (2) denotes substitutive nomenclature. Subsequent names should be considered trivial names frequently encountered in literature.

Table 3. Examples of non-radical reactive oxygen species. Products of The oxidation of organic and inorganic substances acting as prooxidants are shown.

The presence of hydrogen peroxide in Tissues and Cytoplasm is of great importance in oxidative processes. Hydrogen peroxide is formed during the dismutation (interaction) of superoxide anion radicals O₂^•- in the mitochondrial matrix and cell Cytosol, facilitated by the enzyme superoxide dismutase (SOD). The reaction can be written as follows:

2O₂^•- —> 2Н+ —> Н₂О₂ + О₂.

Subsequently, the resulting reaction components can participate in the formation of an even more potent prooxidant—the hydroxyl radical (OH^•)—via the following reaction:

О₂ + Н₂О₂ —> О₂ + ОН^- + ОН^•.

Iron ions can significantly accelerate this process. One of the best-known examples of iron-mediated radical formation is the Fenton reaction, named after Henry Fenton, who first discovered the unusual Features of the interaction between hydrogen peroxide and iron ions in 1894. A more detailed description of this process and the hypothesis regarding free radical formation were put forward half a century later. It was not until the 1950s and 1960s that Changes in the oxidation state of iron and certain other transition metals in reactions involving hydrogen peroxide were discovered. Furthermore, the Biological Significance of processes known as Fenton chemistry was investigated. In the literature, the term "Fenton reaction" is also frequently used. In these processes, iron undergoes oxidation and reduction in the presence of hydrogen peroxide:

(1) Fe²+ + H₂O₂—> Fe³⁺+ HO^• + OH^-,

(2) Fe³+ + О2•- —> Fe²+ + O₂,

overall process: Н₂О₂ + О2•- —> HO• + O₂.

Reaction (1) was first described in a paper by Haber and Weiss, where it was designated as the "Fenton reaction." The hydroxyl radical (HO^•) generated from the cleavage of hydrogen peroxide takes part in numerous oxidation processes involving organic substances—including proteins and Nucleic Acids, but predominantly Lipids. These reactions yield new active radicals and peroxides, which can trigger a chain reaction mechanism and lead to an avalanche-like escalation of processes. This is due to the high reactivity of the hydroxyl radical compared to other prooxidants (Table 4).

Table 4. Reactive oxygen species and related compounds.

Despite significant differences in The activity of the prooxidants presented in the table, all of them can exert a substantial impact on cellular metabolism, induce oxidative stress, and even cause cell death if defense mechanisms fail to operate with sufficient efficiency. It must be noted that the data presented in Table 4 are relative, because assessing the true activity of these substances within a cell also requires considering their half-life ("lifetime") and cytoplasmic concentration. Moreover, prooxidant activity can vary considerably depending on the polarity of the medium. For instance, the activity of O₂•- is low in an aqueous environment but increases significantly in nonpolar media, such as the hydrophobic region of The Lipid Bilayer.

In living cells, various processes contribute to the formation of the hydroxyl radical, which is the most potent oxidizing agent. In addition to iron oxidation processes in the presence of hydrogen peroxide, it is also formed via an analogous copper oxidation process, as well as through the decomposition of peroxynitrite. Furthermore, cells contain a significant amount of hydrogen peroxide generated during mitochondrial Respiration. Meanwhile, Iron and copper metals remain predominantly in a reduced state in the cytoplasm due to the presence of reducing agents such as NADH, NADPH, and others. The cellular production of the hydroxyl radical proceeds through a chain of cyclic reactions (Fig. 41).

Fig. 41. The cyclic process of oxidation and reduction of iron ions by intracellular reducing agents (NADH) and oxidizing agents (Н₂O₂) in the Fenton reaction leads to the generation of hydroxyl radicals, which can cause oxidative damage to Membrane Lipids, proteins, and nucleic acids.

2.2.3. Induced Lipid Oxidation

Three mechanisms of induced lipid oxidation are known: radical autooxidation, photooxidation, and enzymatic oxidation. In living cells, radical processes and reactions participate in such vital processes as electron transport in enzyme respiratory chains and Lipid Metabolism, including the synthesis of Eicosanoids—a group that comprises important substances such as Leukotrienes, Prostaglandins, thromboxanes, and catecholamines. These substances regulate cell proliferation and differentiation, endocytosis and secretion, apoptosis, and cell death. The intensity of Lipid Peroxidation (LPO) depends on the balance between prooxidant and antioxidant activity in the cell. Paradoxically, reducing agents—readily oxidizable substances—can act as the most potent prooxidants capable of inducing free radical formation. These include NADPH₂ and NADH₂, polyunsaturated Fatty acids, and the metabolic products of prostaglandins and catecholamines. Vitamins, such as A and D, can also act as prooxidants if present in excess.

The superoxide anion radical (O₂^•-) plays a crucial role in the oxidation of membrane lipids because its chemical activity increases substantially in the nonpolar environment of the phospholipid bilayer. The superoxide anion radical is capable of attacking the ester bond, resulting in the cleavage of a fatty acid. The peroxyl radical (HO₂^•) possesses even greater activity; moreover, it is uncharged and therefore dissolves more readily in the hydrophobic region of the lipid bilayer than the superoxide anion radical.

One of the most striking examples of the destructive action of lipid oxidation processes is the so-called "respiratory burst"—a sharp increase in oxygen consumption by neutrophils and macrophages during the Immune Response. In this process, oxygen participates in The production of the superoxide anion radical. As a result of subsequent reactions, active oxidizing agents such as hypochlorite and peroxynitrite accumulate in the cytoplasm; these are potent cytotoxic agents that take part in the immune response against bacterial, fungal, and helminthic infections, as well as in The Development of autoimmune diseases. Notably, during the oxidation of hydrocarbon chains in unsaturated lipids, fatty acids can also be cleaved as a result of the attack on ester bonds by phospholipase A₂. This leads to a substantial disruption of membrane barrier properties, which may cause cell death.

The damaging effect of peroxidation processes on cell membranes is associated with the depletion of polyunsaturated lipids within the membranes and the accumulation of oxidation products in the bilayer, including free fatty acids, their oxidized derivatives, and oligomers formed via cross-linking. Furthermore, the accumulation of low-molecular-weight toxic oxidation products, such as aldehydes and dialdehydes (e.g., malondialdehyde), can form cross-links in PROTEIN AND NUCLEIC acid chains.

There are three radical-mediated Stages of Lipid peroxidation (LPO): initiation, propagation, and termination. Let us examine them in detail.

2.2.3.1. LPO Initiation Stage

If hydrogen peroxide is absent from the medium, the LPO initiation process begins with the abstraction of a hydrogen atom from a methylene group (-CH₂-) in the hydrocarbon chain. This process can occur in polyunsaturated fatty acids under the action of a hydroxyl radical (OH^•), whereas the superoxide anion radical O₂•- is insufficiently active for this task. The reaction proceeds According to the formula: -CH₂^- + OH^• —> -CH^• + H₂O.

The LPO process can even be observed in an anaerobic environment if sufficiently active radicals are present (Fig. 42). The presence of a double bond in the chain weakens the C-H bond of the carbon atom adjacent to the double bond, thereby facilitating radical attack and hydrogen abstraction. During the reaction, THE POSITION OF the double bonds shifts, causing adjacent double bonds to approach each other and form a conjugated diene. The final product of this oxidation stage is an alkyl radical. In the presence of singlet oxygen, further oxidation of the hydrocarbon chain occurs, resulting in the formation of a peroxyl radical.

Fig. 42. The LPO process in polyunsaturated Hydrocarbons, such as the fatty acids of biological membrane Phospholipids. In the absence of oxygen, the following occur:

(a) attack of the hydrocarbon chain by free radicals, (b) reorganization of the hydrocarbon molecule, (c) formation of an alkyl radical and conjugation of double bonds with the formation of diene conjugates. In the presence of singlet oxygen: (d) oxidation of the alkyl radical to an alkylperoxyl radical.

2.2.3.2. LPO Propagation Stage

Alkylperoxyl radicals can also actively interact with a neighboring hydrocarbon chain—especially that of polyunsaturated lipids—and Abstract a hydrogen atom.

As a result of the reaction, an inactive alkyl hydroperoxide and an alkyl radical are formed, which is capable of reacting with oxygen again to form an alkylperoxyl radical. Thus, the reaction repeats multiple times, and the concentration of peroxides increases.

2.2.3.3. LPO Termination Stage

Termination (the chain-breaking of oxidation reactions) can occur if the formation of an alkyl radical is prevented. Such reactions can be initiated, for example, in the presence of α-tocopherol, which acts as the primary chain-breaking antioxidant in membranes. Thus, an alkyl radical R• can interact with a peroxyl radical ROO• to form a stable bridge between two hydrocarbon chains (ROOR), or peroxide molecules, by interacting with flavonoids, can form an inactive hydroxylated derivative ROH, as will be shown below.

2.2.4. Other Oxidation Products

2.2.4.1. Formation of Epoxides

Another important class of compounds formed during LPO is epoxides. Epoxides are generated when a double bond is attacked by an alkylperoxyl radical:

Epoxides are detected in biological tissues subjected to oxidative stress, such as following burn injuries or ischemia. Fatty acid epoxides exhibit high biological activity. For instance, linoleic acid epoxides are toxic and are termed leukotoxins (Fig. 43).

Fig. 43. Biologically active epoxides formed during lipid peroxidation. Leukotoxins: (a) 12,13-epoxy-9-octadecenoic acid,

(b) 9,10-epoxy-12-octadecenoic acid, (c) arachidonic acid oxidation product: 5,6-epoxy-8,11,14-eicosatrienoic acid (EET).

The arachidonic acid oxidation product, epoxyeicosatrienoic acid (EET), possesses anti-inflammatory properties and can regulate Tyrosine kinase activity. It participates in calcium signaling and is capable of modulating Blood flow velocity, platelet aggregation, cell migration, apoptosis, Fibrinolysis, and steroid hormone secretion. EET is produced by numerous tissues, including the Liver, Kidneys, Pancreas, Ovaries, as well as endothelial and Brain cells.

2.2.4.2. Cholesterol Oxidation

Depending on the oxidation conditions and the Chemical Nature of the oxidizing agent, Various Forms of oxidized cholesterol can appear (Fig. 44). The oxidation of cholesterol esters depends on the presence of various types of oxidized fatty acids. A detailed description of steroid oxidation processes, chemical structure, and the biological activity of oxidized cholesterol products can be found in Schroepfer's review. An analysis of phytosterol oxidation products can be found in Houvenaghel's review.

Fig. 44. Examples of cholesterol oxidation products: (a) unoxidized cholesterol, (b) 7α-OH or 7β-OH cholesterol, (c) 7-keto cholesterol, (d) 5,6α- or 5,6β-epoxycholesterol.

Many of the most important oxysterols are produced in the cell with the participation of cytochrome P450 complex proteins. For humans, the most important are 27-, 24-, 7α-, and 4β-OH-hydrocholesterols. Similar products can also be formed during the thermal Processing of food during cooking as a result of the so-called autooxidation of cholesterol. The most important autooxidation products are 7-keto- and 7β-OH-cholesterols. Cholesterol oxidation products can exhibit cytotoxic effects, influence Gene Expression, and affect cellular signaling processes.

2.2.4.3. Advanced Lipoxidation End Products

During lipid peroxidation (LPO), fragmentation of polyunsaturated hydrocarbon chains occurs, which can lead to the formation of small, chemically active molecules known as advanced lipoxidation end products (Fig. 45). These include aldehydes such as acrolein, crotonaldehyde, hexanal, malondialdehyde, and glyoxal, as well as 4-hydroxy-2-nonenal (HNE) and other substances.

Fig. 45. Advanced oxidation End products of polyunsaturated lipids: malondialdehyde (a); glyoxal (b); 4-hydroxynonenal or 4-hydroxy-2-nonenal (c).

The best-known and longest-studied is malondialdehyde (MDA), which is formed in many processes of natural lipid peroxidation (Fig. 46). Measuring MDA content is often used to assess the intensity of oxidative processes in tissues. However, the exact mechanism of MDA formation has not been established. It is possible that MDA precursors are monocyclic peroxides formed during the Oxidation of Fatty acids (Fig. 46). Prostaglandin precursors can also serve as a source of MDA. For instance, thromboxane synthase can generate MDA alongside thromboxane A2 by acting on prostaglandin endoperoxide during the activation of human Blood Platelets. MDA is a very important LPO product because it is highly reactive and can interact with proteins and polynucleotides, forming cross-links between chains. Aldehydes are capable of interacting with various molecules, including proteins, Amino Acids, and aminophospholipids, resulting in significant changes in their properties, which leads to cell death and tissue damage. The products of the chemical interaction of these agents with the molecular Components of the cell are called adducts (Fig. 47).

Fig. 46. Proposed process of MDA formation. An alkylperoxyl radical (a) interacts with a neighboring double bond located on the same chain, resulting in the formation of a monocyclic peroxide, the rearrangement of double bonds, and the acquisition of an unpaired electron by the adjacent carbon atom (b). During the further oxidation of the resulting alkylperoxyl radical, an alkylhydroperoxyl is formed, followed by the Cleavage of the hydrocarbon chain (c) and the formation of MDA (d).

Fig. 47. Damage to aminophospholipids, proteins, and DNA by aldehyde products of lipid peroxidation. As an example, molecular adducts (advanced peroxidation end products) formed upon interaction with the dialdehyde glyoxal are shown. The resulting terminal carboxyl groups are capable of entering into further interactions, forming cross-links between molecules.

2.2.5. Flavonoids as Antioxidants

The antioxidant properties of flavonoids are widely recognized. Many hypotheses regarding The impact of flavonoids on human health, including positive effects on The Cardiovascular system, anti-carcinogenic activity, etc., are also based on their antioxidant properties. Flavonoids, along with other dietary antioxidants such as vitamins E and C, are vital components of the cellular antioxidant system. In accordance with the generally accepted view, the antioxidant properties of flavonoids are based on their ability to act as free radical scavengers and to chelate metal ions involved in peroxidation.

Polyphenolic compounds (Phn) are capable of interacting with hydroxyl (L-O^•) and lipid peroxyl (L-OO^•) radicals (alkoxyls) due to their ability to donate an electron (or hydrogen atom). As a result, phenol radicals—phenoxyls—are formed, which do not participate in the Propagation of the oxidative process. This is due to the unique structure of their molecule, in which the stabilization of the electron cloud occurs:

(1) L-OO^• + Phn-OH —> L-OOH + Phn-O^•

(2) L-O^• + Phn-OH —> L-OH + Phn-O^•

Furthermore, the reduction of oxidized polyphenols is possible and can occur via various pathways, for example, with the participation of ascorbic acid (Asc). As a result, a monodehydroascorbate radical (MDA^•) is formed, which is converted into ascorbic acid and dehydroascorbic acid (DHA):

(1) 2Phn-O^• + Asc —> 2Phn-OH + 2MDA^•

(2) MDA^• + MDA^• —> Asc + DHA.

Flavonoid molecules contain three regions that are primarily responsible for radical-scavenging properties (Fig. 48): (1) a group of two adjacent hydroxyls on the B-ring, termed the catechol group; (2) a 2,3-double bond conjugated with the 4-oxo group, which is presumably capable of initiating electron delocalization in the B-ring; (3) hydroxyl groups at positions 3 and 5, which scavenge radicals.

Fig. 48. Quercetin molecule. The groups primarily responsible for free radical scavenging are indicated.

It is hypothesized that the hydroxyls of the catechol group of the B-ring or the hydroxyl group at the C-3 position serve as the primary target for various oxidants. Upon oxidation of these groups, short-lived semiquinone radical anions are formed first, followed by ortho-Quinones. It is suggested that these hydroxyl groups may be involved in a unified process of intramolecular transformations (Fig. 49).

Fig. 49. Proposed pathway of molecular transformations of quercetin initiated by radical attack on the catechol group of ring B.

As shown in Table 5, quercetin and myricetin exhibit a rather high activity because they contain virtually all structural elements responsible for radical-scavenging capacity. Overall, flavan-3-ols are even more active, especially epigallocatechin gallate (EGCG). It should be noted that flavan-3-ols—i.e., flavonoids bearing a hydroxyl group at position 3, which is crucial for maintaining antiradical activity—gain additional potency if they also possess a catechol group. Conversely, flavonoids with a catechol group and a 2,3-double bond, but lacking a 3-hydroxyl group, show reduced activity. This highlights the requirement for a combination of all these structural factors to achieve high activity.

Table 5. Structure-activity relationship of flavonoids in the presence of the diphenylpicrylhydrazyl radical.

Note. EC - epicatechin, EGC - epigallocatechin, ECG - epicatechin gallate, EGCG - epigallocatechin gallate.

An important factor determining the antioxidant activity of flavonoids toward the hydrocarbon chains of the lipid bilayer is their Hydrophobicity. For instance, EGCG has been shown to be highly hydrophobic and readily dissolves within the bilayer. This agent is more hydrophobic than vitamin E, which accounts for its superior efficacy in protecting the lipid bilayer. The simplified Overview of flavonoid antioxidant properties presented above would be incomplete without a broader analysis of their free radical-scavenging capabilities. To compare the free radical-trapping efficiencies of flavonoids, pulse radiolysis can be employed to measure the reaction rates between flavonoids and various radicals. It has been found that the radical-scavenging properties of flavonoids vary significantly depending on the specific reactive agent. Thus, catechin gallates exhibit high activity exclusively against the superoxide radical (O₂•-), but show little effect on other, often much more reactive radicals. Conversely, luteolin and kaempferol—which display low activity toward the superoxide radical—show the highest activity against OH^•, N₃^•, and t-BuO^• radicals. Furthermore, the ability of quercetin to protect against autoxidation processes in rat brain membrane fractions appears rather modest when compared to the flavonoids naringenin and hesperetin.

Theoretical studies indicate that the antioxidant activity of catechins is related to the ability of the catechol hydroxyl groups to form Hydrogen Bonds with two oxygen atoms of the lipid peroxyl radical, resulting in radical trapping and the formation of a compact reactive complex.

Currently, no universal theory bridges the relationship between flavonoid structure and antioxidant activity; however, it can be hypothesized that different flavonoids specialize in protecting the organism from various damaging agents. It is incorrect to claim that certain flavonoids are more effective than others without considering specific experimental conditions and The Nature of the oxidizing agent. The vast diversity of flavonoids in nature allows these compounds to provide comprehensive protection for organisms against a multitude of environmental threats.

2.2.6. Oxidation products in cellular signaling

Normal physiological processes in cells involve the production of the superoxide radical O₂•^- mediated by NADPH oxidases, which are present in all cell types. In addition, O₂^•- is generated by mitochondria, which contain several distinct superoxide production sites. These include the ubiquinone-binding sites of complex I (site IQ) and complex III (site IIIQo), glycerol-3-phosphate dehydrogenase, flavin complex I (site IF), electron-transferring flavoprotein Q-oxidoreductase (ETFQOR) of fatty acid β-oxidation, cytochrome c oxidoreductase, the cytochrome bc1 complex, and finally, Pyruvate and 2-oxoglutarate dehydrogenases. The potential for damage to the mitochondrial matrix or DNA depends on the localization of these sites. While all these sites are located in the matrix, site IIIQo and glycerol-3-phosphate dehydrogenase are also found in the intermembrane space, which facilitates the release of superoxide into the cytoplasm. The relative contribution of these sites to superoxide production largely depends on the cell type and can vary under different physiological conditions. Mitochondrial superoxide dismutase can neutralize the generated superoxide radical—the primary component of mitochondrial reactive oxygen species (ROS)—which regulates cell survival or death pathways, modulates retrograde signaling from the cytoplasm to The Nucleus, and participates in numerous physiological processes (Fig. 50).

Fig. 50. Cellular localization of the Sites of production and propagation of the superoxide radical and hydrogen peroxide. Superoxide is produced extracellularly by NADPH oxidase located on The Plasma Membrane (1), then dismutates via extracellular superoxide dismutases to form H₂О₂ (2), which can enter the cytoplasm through Aquaporins (3). Superoxide can also enter the cell through anion channels (4), where it is dismutated into H₂О₂ and О₂ by Cu- and Zn-dependent superoxide dismutases (Cu/ZnSOD) (5). In the cytoplasm, peroxiredoxins Prdx (6) and glutathione peroxidases Gpx (7) rapidly scavenge H₂О₂. These reactions are coupled with the oxidation of glutathione (GSH) and thioredoxin Trx(SH)₂, yielding glutathione disulfide (GSSG) and thioredoxin disulfide (TrxS₂). Catalase present in Peroxisomes also removes excess H₂О₂ (8). Superoxide is produced by mitochondria through the oxidation of ubisemiquinone (9) via a reaction initiated by Mn-dependent superoxide dismutase (MnSOD) (10), resulting in the formation of H₂О₂. If O₂•- is generated on the outer surface of The inner mitochondrial membrane, it dismutates via Cu/Zn SOD (11), yielding H₂О₂ and О₂. NADPH oxidase present on the outer nuclear membrane or Endoplasmic reticulum membranes produces О₂^•- (12). Further details can be found in the review.

This mechanism of signal propagation between mitochondria, other Organelles, and the cellular environment is targeted by numerous pharmacological agents. Upon entering the cellular milieu, superoxide can cross into the cytoplasm of neighboring cells via anion channels and participate in signaling cascades. However, once in the cytoplasm, it can be rapidly eliminated by superoxide dismutase, which is present in high concentrations and exhibits remarkably high catalytic activity, yielding hydrogen peroxide and Water. Consequently, the actual contribution of the superoxide radical to Intercellular Communication remains an open question.

Unlike the superoxide radical, the role of hydrogen peroxide as a secondary messenger is well established. The superoxide radical serves as its direct and primary precursor owing to mitochondrial superoxide dismutase activity. An additional source of hydrogen peroxide can be provided by two Nox/Duox family NADPH oxidases, which catalyze the two-electron reduction of oxygen in phagocytes, thereby ensuring the bactericidal action of these cells. Hydrogen peroxide is also utilized to signal cell growth during angiogenesis, in the development of immune responses, in cellular adaptation to Hypoxia, and in the oxidative modification of Extracellular matrix proteins. Due to these functions, Nox/Duox family NADPH oxidases serve as therapeutic targets for the Treatment of Cancer, inflammatory conditions, neurodegenerative disorders, and as regulators of cardiomyocyte and pulmonary epithelial cell growth.

Another key regulator affecting post-translational modification signaling pathways is 4-hydroxynonenal (4-HNE), a lipid peroxidation product of polyunsaturated fatty acids. This compound is detected in all animal tissues upon the activation of oxidative stress. It is hypothesized that 4-HNE may play a pivotal role in mediating Cell Division and Cell Adhesion signals.

The presence of 4-HNE in tissues can trigger inflammatory, neurodegenerative, respiratory, and oncological diseases. Intracellular levels of 4-HNE are regulated by the activity of glutathione S-transferases (GSTs), which conjugate glutathione-containing Peptides with 4-HNE to form water-soluble GS-HNE molecules that are incapable of initiating oxidative stress.

Recently, it was discovered that certain flavonoids also exert a protective effect on cells exposed to elevated 4-HNE concentrations. For instance, kaempferol can protect PC-12 cells from 4-HNE-induced apoptosis. Kaempferol interacts directly with the cytoplasmic subunit of NADPH oxidase (the p47phox subunit), thereby inhibiting the aberrant NADPH-HNE-induced activation of NADPH oxidase. This action terminates the signaling cascade running from NADPH oxidase to c-Jun N-terminal kinase (JNK) and subsequently to the Bcl-2 apoptotic proteins and caspase-3 (the NOX-JNK signaling pathway). Consequently, kaempferol shows promise as an effective prophylactic agent against NADPH oxidase-mediated neurodegeneration.

Previously, the same researchers demonstrated that red wine flavonoids and cocoa procyanidins can exert a similar protective effect. Notably, the most active agent was not the well-known red wine compound resveratrol, but rather the flavonoids quercetin and myricetin, which are also present in wine. The authors attributed the ability of these flavonoids to prevent cellular apoptosis to the protection of poly(ADP-ribose) polymerase from the inhibitory action of 4-HNE. As for cocoa procyanidins, their targets included the apoptotic proteins Bcl-2 and Bcl-xL, whose activity was suppressed. Concurrently, an increase in caspase-3 activity, along with the activation of c-Jun N-terminal kinase (JNK) and mitogen-activated protein kinase kinase 4 (MKK4), was observed.

Numerous studies indicate that soy isoflavones also reduce the risk of breast cancer through protection against 4-HNE-induced damage. In particular, genistein has been found to modulate glutathione S-transferase expression in human mammary epithelial cells that had been disrupted by the toxic action of lipid peroxidation products, specifically 4-HNE. This diminishes the potential for cellular DNA Damage caused by 4-HNE, thereby preventing neoplastic tissue growth and tumor development.